Journal: PLoS ONE

Article Title: TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion

doi: 10.1371/journal.pone.0042316

Figure Lengend Snippet: Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), phospho-4EBP-1 (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.

Article Snippet: Phospho-Akt (Ser473), phospho-Akt (thr-308), Akt, phospho-S6 kinase, S6 kinase, phospho-4EBP-1 (Thr-37/46) phospho-4EBP-1 (Ser-65) 4EBP-1, phospho-GSK3β, GSK3β, phospho-tuberin (Thr 1462), tuberin, phospho-PRAS40 (Thr 246), PRAS40, phospho-mTOR (Ser-2448) and mTOR antibodies were purchased from Cell Signaling, Boston, MA. siRNA pool of three oligonucleotides against PTEN mRNA, collagen II (α2) and PTEN antibodies were obtained from Santacruz, Delaware, CA.

Techniques: Transfection, Plasmid Preparation, Incubation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models

doi: 10.1016/j.jcmgh.2021.11.007

Figure Lengend Snippet: TM6SF2 lacking promotes cell survival and carcinogenesis. ( A ) Cell growth was assessed through MTS assay for 24, 48, 72 hours and 1 week (λ = 490 nm). Data are expressed as fold increase vs control. ( B ) Representative images of wound healing assay were acquired at 0, 24, 48 hours (magnification, 100×). The dotted lines indicate the scratch width. ( C ) Phosphorylation of Akt at serine 437 residue (P[S437]-Akt), mTOR at serine 2448 residue (p[S2448]-mTOR), phosphorylation of 4E-BP1 at threonine 37/46 residues P[Thr37/46]-4E-BP1, and total Akt, mTOR, and 4E-BP1 were evaluated by Western blot. ( D–F ) Quantification of P(S437)-Akt/total Akt, p(S2448)-mTOR/total mTOR, and P(Thr37/46)-4E-BP1/total 4E-BP1 ratios were measured through ImageJ software and normalized to the β-actin housekeeping gene. At least 3 independent experiments were conducted. For bar graphs, data are expressed as means and SE. Adjusted ∗ P < .05 and ∗∗ P < .01 vs Cas9 + and/or vs MBOAT7 -/- . AU, arbitrary unit.

Article Snippet: Anti–P-Ser473 Akt, Akt, P(Thr37/46)-4E-BP1, 4E-BP1, P(Ser2448)-mTOR, and mTOR antibodies were acquired from Cell Signaling Technology (Danvers, MA).

Techniques: MTS Assay, Wound Healing Assay, Western Blot, Software

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models

doi: 10.1016/j.jcmgh.2021.11.007

Figure Lengend Snippet: List of Antibodies and Relative Dilutions Used in Western Blot and Immunocytochemistry Experiments

Article Snippet: Anti–P-Ser473 Akt, Akt, P(Thr37/46)-4E-BP1, 4E-BP1, P(Ser2448)-mTOR, and mTOR antibodies were acquired from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Immunocytochemistry

Journal: International Journal of Hepatology

Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

doi: 10.1155/2013/103830

Figure Lengend Snippet: Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

Techniques: Inhibition, MTT Assay, Labeling, Expressing, Western Blot

Journal: International Journal of Hepatology

Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

doi: 10.1155/2013/103830

Figure Lengend Snippet: Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

Techniques: Activity Assay, MTT Assay, Expressing, Western Blot

Journal: International Journal of Hepatology

Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

doi: 10.1155/2013/103830

Figure Lengend Snippet: Everolimus inhibited proliferation and mTOR signaling in HCC cell lines. (a) Dose-dependent inhibition of HCC cell proliferation by everolimus. The effect of everolimus on cell viability was assessed by MTT assay. Dose-response curves of everolimus for all HCC cell lines were shown. Similar results were observed in 3 independent experiments. (b) Average IC 50 values of everolimus in HCC cell lines. Cumulative results from 3 independent experiments were shown as mean ± SEM. (c) Everolimus inhibited the mTOR pathway in HCC cells. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with 0.1 μ M everolimus (hereafter labeled as Eve) or DMSO control for 48 hrs and 72 hrs. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

Techniques: Inhibition, MTT Assay, Labeling, Expressing, Western Blot

Journal: International Journal of Hepatology

Article Title: Enhanced Antitumor Activity with Combining Effect of mTOR Inhibition and Microtubule Stabilization in Hepatocellular Carcinoma

doi: 10.1155/2013/103830

Figure Lengend Snippet: Enhanced antitumor activity of the everolimus/patupilone combination in HCC cell lines. (a) Effects of everolimus/patupilone in HCC cell lines. HepG2, Hep3B, and SNU398 cells (1 × 10 4 ) were treated with various concentrations of everolimus in combination with 0.5 nM patupilone (Pat) for 24 hrs. Cell viability was assessed by MTT assay. Cumulative results from 3 independent experiments were shown as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001 versus everolimus-treated group). (b) The mTOR signaling in HCC cells was not further suppressed by the everolimus/patupilone combination treatment. HepG2, Hep3B, and SNU398 cells (3 × 10 5 ) were treated with everolimus (0.1 μ M) and/or patupilone (Pat) (0.5 nM) for 24 hrs. The everolimus/patupilone combination is abbreviated as Eve/Pat hereafter. The expression levels of the mTOR pathway components, pi-mTOR (ser2448), mTOR, pi-p70S6K (Thr389), p70S6K, pi-S6 (ser240/244), S6, pi-4E-BP1 (ser65), and 4E-BP1, and actin were examined by western blotting. Similar results were observed in 3 independent experiments.

Article Snippet: The following antibodies were used in the study: anti-mTOR, anti-pi-mTOR (ser2448), anti-Akt, anti-pi-Akt (ser473), anti-p70S6k, anti-pi-p70S6k (Thr389), anti-S6, anti-pi-S6 (ser240/244), anti-4E-BP1, anti-pi-4E-BP1 (ser65), anti-cleaved PARP (all from Cell Signaling Technology, Beverly, MA, USA), and anti-actin (Calbiochem, Nottingham, UK).

Techniques: Activity Assay, MTT Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models

doi: 10.1371/journal.pone.0057431

Figure Lengend Snippet: A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, and 4E-BP1 obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 , where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at * P <0.02 in IBC SUM-149 cells. C. 1×10 5 cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[ 35 S] Methionine and L-[ 35 S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (* P <0.05) reduces protein synthesis by 48%.

Article Snippet: We used a monoclonal anti-Phospho-mTOR (Ser2481), monoclonal anti-mTOR, anti-Phospho-4E-BP1 (Thr37/46), anti-Phospho-S6 Ribosomal Protein (Ser235/236), anti-S6 Ribosomal Protein, anti-Akt, anti-Phospho-Akt (Ser473), anti-eIF4G and anti-eIF4E and polyclonal anti-p70s6 kinase (Cell Signaling, Danvers, MA) antibodies.

Techniques: Lysis, Western Blot, Incubation, Radioactivity